DELLA proteins positively regulate seed size in Arabidopsis.

Human and animal nutrition is mainly based on seeds. Seed size is a key factor affecting seed yield and has thus been one of the primary objectives of plant breeders since the domestication of crop plants. Seed size is coordinately regulated by signals of maternal and zygotic tissues that control the growth of the seed coat, endosperm and embryo. Here, we provide previously unreported evidence for the role of DELLA proteins, key repressors of gibberellin responses, in the maternal control of seed size. The gain-of-function della mutant gai-1 produces larger seeds as a result of an increase in the cell number in ovule integuments. This leads to an increase in ovule size and, in turn, to an increase in seed size. Moreover, DELLA activity promotes increased seed size by inducing the transcriptional activation of AINTEGUMENTA, a genetic factor that controls cell proliferation and organ growth, in the ovule integuments of gai-1. Overall, our results indicate that DELLA proteins are involved in the control of seed size and suggest that modulation of the DELLA-dependent pathway could be used to improve crop yield.

Gibberellins regulate ovule number through a DELLA-CUC2 complex in Arabidopsis.

Ovule development is a key process for plant reproduction, helping to ensure correct seed production. Several molecular factors and plant hormones such as gibberellins are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been shown to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA acts in ovule primordia initiation remained unknown. In this study we report that DELLA proteins participate in ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, through the direct GAI-CUC2 interaction in placental cells that would determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and co-localization in the placenta supports this hypothesis. Moreover, molecular analysis identified a subset of the loci for which the GAI protein may act as a transcriptional co-regulator in a CUC2-dependent manner. The DELLA-CUC2 complex is a component of the gene regulatory network controlling ovule primordia initiation in Arabidopsis.

IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like peptides and HAE (HAESA)-like receptors regulate corolla abscission in Nicotiana benthamiana flowers.

BACKGROUND: Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana, abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species. RESULTS: The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana. These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides. CONCLUSION: Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA-like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms.

Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis.

Ovule development is essential for plant survival, as it allows correct embryo and seed development upon fertilization. The female gametophyte is formed in the central area of the nucellus during ovule development, in a complex developmental programme that involves key regulatory genes and the plant hormones auxins and brassinosteroids. Here we provide novel evidence of the role of gibberellins (GAs) in the control of megagametogenesis and embryo sac development, via the GA-dependent degradation of RGA-LIKE1 (RGL1) in the ovule primordia. YPet-rgl1Δ17 plants, which express a dominant version of RGL1, showed reduced fertility, mainly due to altered embryo sac formation that varied from partial to total ablation. YPet-rgl1Δ17 ovules followed normal development of the megaspore mother cell, meiosis, and formation of the functional megaspore, but YPet-rgl1Δ17 plants had impaired mitotic divisions of the functional megaspore. This phenotype is RGL1-specific, as it is not observed in any other dominant mutants of the DELLA proteins. Expression analysis of YPet-rgl1Δ17 coupled to in situ localization of bioactive GAs in ovule primordia led us to propose a mechanism of GA-mediated RGL1 degradation that allows proper embryo sac development. Taken together, our data unravel a novel specific role of GAs in the control of female gametophyte development.

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms.

Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co-regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.

An Improved Recombineering Toolset for Plants.

Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing have made it possible to directly modify the target genes in their native chromosomal locations, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the results obtained by direct chromosomal tagging using genome-editing strategies. These simple, scalable, customizable recombineering toolsets and protocols allow a variety of genetic modifications to be generated. In addition, we developed a highly efficient recombinase-mediated cassette exchange system to facilitate the transfer of the desired sequences from a bacterial artificial chromosome clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis (Arabidopsis thaliana). We demonstrated the utility of this system by generating more than 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes and characterizing the translational reporter expression patterns for 14 auxin biosynthesis genes.

RGL2 controls flower development, ovule number and fertility in Arabidopsis.

DELLA proteins are a group of plant specific GRAS proteins of transcriptional regulators that have a key role in gibberellin (GA) signaling. In Arabidopsis, the DELLA family is formed by five members. The complexity of this gene family raises the question on whether single DELLA proteins have specific or overlapping functions in the control of several GA-dependent developmental processes. To better understand the roles played by RGL2, one of the DELLA proteins in Arabidopsis, two transgenic lines that express fusion proteins of Venus-RGL2 and a dominant version of RGL2, YPet-rgl2Δ17, were generated by recombineering strategy using a genomic clone that contained the RGL2 gene. The dominant YPet-rgl2Δ17 protein is not degraded by GAs, and therefore it blocks the RGL2-dependent GA signaling and hence RGL2-dependent development. The RGL2 role in seed germination was further confirmed using these genetic tools, while new functions of RGL2 in plant development were uncovered. RGL2 has a clear function in the regulation of flower development, particularly stamen growth and anther dehiscence, which has a great impact in fertility. Moreover, the increased ovule number in the YPet-rgl2Δ17 line points out the role of RGL2 in the determination of ovule number.

Arabidopsis Heat Stress-Induced Proteins Are Enriched in Electrostatically Charged Amino Acids and Intrinsically Disordered Regions.

Comparison of the proteins of thermophilic, mesophilic, and psychrophilic prokaryotes has revealed several features characteristic to proteins adapted to high temperatures, which increase their thermostability. These characteristics include a profusion of disulfide bonds, salt bridges, hydrogen bonds, and hydrophobic interactions, and a depletion in intrinsically disordered regions. It is unclear, however, whether such differences can also be observed in eukaryotic proteins or when comparing proteins that are adapted to temperatures that are more subtly different. When an organism is exposed to high temperatures, a subset of its proteins is overexpressed (heat-induced proteins), whereas others are either repressed (heat-repressed proteins) or remain unaffected. Here, we determine the expression levels of all genes in the eukaryotic model system Arabidopsis thaliana at 22 and 37 °C, and compare both the amino acid compositions and levels of intrinsic disorder of heat-induced and heat-repressed proteins. We show that, compared to heat-repressed proteins, heat-induced proteins are enriched in electrostatically charged amino acids and depleted in polar amino acids, mirroring thermophile proteins. However, in contrast with thermophile proteins, heat-induced proteins are enriched in intrinsically disordered regions, and depleted in hydrophobic amino acids. Our results indicate that temperature adaptation at the level of amino acid composition and intrinsic disorder can be observed not only in proteins of thermophilic organisms, but also in eukaryotic heat-induced proteins; the underlying adaptation pathways, however, are similar but not the same.

Gibberellins negatively modulate ovule number in plants.

Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.

Bud sprouting and floral induction and expression of FT in loquat [Eriobotrya japonica (Thunb.) Lindl.].

MAIN CONCLUSION: EjFT1 and EjFT2 genes were isolated and sequenced from leaves of loquat. EjFT1 is involved in bud sprouting and leaf development, and EjFT2 in floral bud induction. Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an evergreen species belonging to the family Rosaceae, such as apple and pear, whose reproductive development, in contrast with these species, is a continuous process that is not interrupted by winter dormancy. Thus, the study of the mechanism of flowering in loquat has the potential to uncover the environmental and genetic networks that trigger flowering more accurately, contributing for a better understanding of the Rosaceae floral process. As a first step toward understanding the molecular mechanisms controlling flowering, extensive defoliation and defruiting assays, together with molecular studies of the key FLOWERING LOCUS T (FT) gene, were carried out. FT exhibited two peaks of expression in leaves, the first one in early to mid-May, the second one in mid-June. Two FT genes, EjFT1 and EjFT2, were isolated and sequenced and studied their expression. Expression of EjFT1 and EjFT2 peaks in mid-May, at bud sprouting. EjFT2 expression peaks again in mid-June, coinciding with the floral bud inductive period. Thus, when all leaves of the tree were continuously removed from early to late May vegetative apex differentiated into panicle, but when defoliation was performed from early to late June apex did not differentiate. On the other hand, fruit removal advanced EjFT1 expression in old leaves and the sooner the fruit detached, the sooner the bud sprouted. Accordingly, results strongly suggest that EjFT1 might be related to bud sprouting and leaf development, while EjFT2 might be involved in floral bud induction. An integrative model for FT functions in loquat is discussed.

Corrigendum: Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus.

[This corrects the article on p. 126 in vol. 8, PMID: 28228766.].

Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus.

Abscission is a cell separation process by which plants can shed organs such as fruits, leaves, or flowers. The process takes place in specific locations termed abscission zones. In fruit crops like citrus, fruit abscission represents a high percentage of annual yield losses. Thus, understanding the molecular regulation of abscission is of capital relevance to control production. To identify genes preferentially expressed within the citrus fruit abscission zone (AZ-C), we performed a comparative transcriptomics assay at the cell type resolution level between the AZ-C and adjacent fruit rind cells (non-abscising tissue) during ethylene-promoted abscission. Our strategy combined laser microdissection with microarray analysis. Cell wall modification-related gene families displayed prominent representation in the AZ-C. Phylogenetic analyses of such gene families revealed a link between phylogenetic proximity and expression pattern during abscission suggesting highly conserved roles for specific members of these families in abscission. Our transcriptomic data was validated with (and strongly supported by) a parallel approach consisting on anatomical, histochemical and biochemical analyses on the AZ-C during fruit abscission. Our work identifies genes potentially involved in organ abscission and provides relevant data for future biotechnology approaches aimed at controlling such crucial process for citrus yield.

Gibberellins Regulate Ovule Integument Development by Interfering with the Transcription Factor ATS.

Gibberellins (GAs) are plant hormones that regulate most plant life cycle aspects, including flowering and fruit development. Here, we demonstrate the implication of GAs in ovule development. DELLA proteins, negative GA response regulators, act as positive factors for ovule integument development in a mechanism that involves transcription factor ABERRANT TESTA SHAPE (ATS). The seeds of the della global mutant, a complete loss-of-function of DELLA, and the ats-1 mutant are remarkably similar, with a round shape, a disorganized testa, and viviparism. These defects are the result of an alteration in integuments that fail to fully develop and are shorter than in wild-type plants. ats-1 also shows some GA-related phenotypes, for example, higher germination rates and early flowering. In fact, ats-1 has elevated GA levels due to the activation of GA biosynthesis genes, which indicates that ATS inhibits GA biosynthesis. Moreover, DELLAs and ATS proteins interact, which suggests the formation of a transcriptional complex that regulates the expression of genes involved in integument growth. Therefore, the repression of GA biosynthesis by ATS would result in the stabilization of DELLAs to ensure correct ATS-DELLA complex formation. The requirement of both activities to coordinate proper ovule development strongly argues that the ATS-DELLA complex acts as a key molecular factor. This work provides the first evidence for a role of GAs in ovule and seed development.

The IDA Peptide Controls Abscission in Arabidopsis and Citrus.

Organ abscission is an important process in plant development and reproduction. During abscission, changes in cellular adhesion of specialized abscission zone cells ensure the detachment of infected organs or those no longer serving a function to the plant. In addition, abscission also plays an important role in the release of ripe fruits. Different plant species display distinct patterns and timing of organ shedding, most likely adapted during evolution to their diverse life styles. However, it appears that key regulators of cell separation may have conserved function in different plant species. Here, we investigate the functional conservation of the citrus ortholog of the Arabidopsis peptide ligand INFLORESCENCE DEFICIENT IN ABSCISSION (AtIDA), controlling floral organ abscission. We discuss the possible implications of modifying the citrus IDA ortholog for citrus fruit production.

Role of the gibberellin receptors GID1 during fruit-set in Arabidopsis.

Gibberellins (GAs) play a critical role in fruit-set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit-set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue-specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C-RGL1 and GID1B-RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1-DELLA in the different GA-dependent processes that occur upon fruit-set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.

Molecular programme of senescence in dry and fleshy fruits.

Fruits of angiosperms can be divided into dry and fleshy fruits, depending on their dispersal strategies. Despite their apparently different developmental programmes, researchers have attempted to compare dry and fleshy fruits to establish analogies of the distinct biochemical and physiological processes that occur. But what are the common and specific phenomena in both biological strategies? Is valve dehiscence and senescence of dry fruits comparable to final ripening of fleshy fruits, when seeds become mature and fruits are competent for seed dispersal, or to over-ripening when advanced senescence occurs? We briefly review current knowledge on dry and fleshy fruit development, which has been extensively reported recently, and is the topic of this special issue. We compare the processes taking place in Arabidopsis (dry) and tomato (fleshy) fruit during final development steps using transcriptome data to establish possible analogies. Interestingly, the transcriptomic programme of Arabidopsis silique shares little similarity in gene number to tomato fruit ripening or over-ripening. In contrast, the biological processes carried out by these common genes from ripening and over-ripening programmes are similar, as most biological processes are shared during both programmes. On the other hand, several biological terms are specific of Arabidopsis and tomato ripening, including senescence, but little or no specific processes occur during Arabidopsis and tomato over-ripening. These suggest a closer analogy between silique senescence and ripening than over-ripening, but a major common biological programme between Arabidopsis silique senescence and the last steps of tomato development, irrespective of its distinction between ripening and over-ripening.

Systems analysis of auxin transport in the Arabidopsis root apex.

Auxin is a key regulator of plant growth and development. Within the root tip, auxin distribution plays a crucial role specifying developmental zones and coordinating tropic responses. Determining how the organ-scale auxin pattern is regulated at the cellular scale is essential to understanding how these processes are controlled. In this study, we developed an auxin transport model based on actual root cell geometries and carrier subcellular localizations. We tested model predictions using the DII-VENUS auxin sensor in conjunction with state-of-the-art segmentation tools. Our study revealed that auxin efflux carriers alone cannot create the pattern of auxin distribution at the root tip and that AUX1/LAX influx carriers are also required. We observed that AUX1 in lateral root cap (LRC) and elongating epidermal cells greatly enhance auxin's shootward flux, with this flux being predominantly through the LRC, entering the epidermal cells only as they enter the elongation zone. We conclude that the nonpolar AUX1/LAX influx carriers control which tissues have high auxin levels, whereas the polar PIN carriers control the direction of auxin transport within these tissues.

Comparison of global responses to mild deficiency and excess copper levels in Arabidopsis seedlings.

Copper is an essential micronutrient in higher plants, but it is toxic in excess. The fine adjustments required to fit copper nutritional demands for optimal growth are illustrated by the diverse, severe symptoms resulting from copper deficiency and excess. Here, a differential transcriptomic analysis was done between Arabidopsis thaliana plants suffering from mild copper deficiency and those with a slight copper excess. The effects on the genes encoding cuproproteins or copper homeostasis factors were included in a CuAt database, which was organised to collect additional information and connections to other databases. The categories overrepresented under copper deficiency and copper excess conditions are discussed. Different members of the categories overrepresented under copper deficiency conditions were both dependent and independent of the general copper deficiency transcriptional regulator SPL7. The putative regulatory elements in the promoter of the copper deficiency overrepresented genes, particularly of the iron superoxide dismutase gene FSD1, were also analysed. A 65 base pair promoter fragment, with at least three GTAC sequences, was found to be not only characteristic of them all, but was responsible for most of the FSD1 copper-dependent regulations. Moreover, a new molecular marker for the slight excess copper nutritional status is proposed. Taken together, these data further contribute to characterise copper nutritional responses in higher plants.

Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling.

Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes, including respiration, photosynthesis, and oxidative stress protection. In many eukaryotic organisms, including yeast (Saccharomyces cerevisiae) and mammals, copper and iron homeostases are highly interconnected; yet, such interdependence is not well established in higher plants. Here, we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis (Arabidopsis thaliana). COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 could play a dual role under iron deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation, possibly to minimize further iron consumption. Second, global expression analyses of copt2-1 versus wild-type Arabidopsis plants indicate that low-phosphate responses increase in the mutant. These results open up new biotechnological approaches to fight iron deficiency in crops.

AUX/LAX genes encode a family of auxin influx transporters that perform distinct functions during Arabidopsis development.

Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms.